The detection of pathogens is critical to the prevention and identification of problems related to health and safety. It is important to obtain analytical results in the shortest time possible. This is not always possible with traditional pathogen detection methods. The advent of new technologies including the use of biosensors has led to promising results.
Various pathogens are known that are potentially harmful to humans. These pathogens include but are not limited to the following that are studied in this disclosure: Pseudomonas aeruginosa, Staphyloccocus aureus, Escherichia coli, Porphyromonas gingivalis, Listeria monocytogens. Below is background information for these pathogens in relation to health and safety.
Pseudomonas aeruginosa (P. aeruginosa)
Pseudomonas aeruginosa (P. aeruginosa) is an aerobic non-fermenting, motile, gram negative rod commonly found free in moist environment, in human skin and gut as a normal flora (Warburton D W et al., 1994). However, P. aeruginosa is considered one of the most common pathogens of plant, animal and human (Sherris J C, 1990). It can be postulated in the pathogenesis of many nosocomial pneumonia, urinary tract infection, wound infection and gram-negative bacteremia (National Nosocomial Infection Surveillance (NNIS) system report, 1992-2003). Serious morbidity and mortality were reported in 18-61% of severe pseudomonas infections, mainly attributed to a delay in the diagnosis and inappropriate treatment (Fujitani S et al., 2011). P. aeruginosa infection is considered the commonest pathogen infecting the airways of cystic fibrosis patients (Cystic Fibrosis Foundation Annual Patient Registry 2010). P. aeruginosa has panoply of virulent factors including toxins and surface components (Somerville G, 1999), in both animals and humans, two modes of virulence expression, and two different pathogenetic performance. One chronic, indolent colonization and another acute invasion with associated septic shock and death especially in immunocompromised hosts. Chronic P. aeruginosa infection in patients with cystic fibrosis is associated with an expression of a mucoid phenotype together with down regulation of virulent factors (Zhao J, 2012). Acute invasive P. aeruginosa infections is mediated through exotoxins (A, S, and U), elastase (LasA and Lasb), alkaline protease, cytotoxins, phospholipase C, phenazines and cell bound organelles (pili, flagella, and membrane bound lipopolysaccharide) (Mahajan-Miklos S, 1999). Antibiotic resistance by P. aeruginosa develops through expression of a beta-lactamase or efflux pumps and down regulation of outer membrane porins (Anderson D J et al., 2012).
Specific detection of proteases secreted by the pathogenic organism under control als rhl qurorum sensing, is a promising target for the analysis of wound infection. Protease based detection methods for diagnosing bacterial infection has been previously described (Sun H et al., 2012; Rittich B et al., 2006). It was founded as most sensitive and specific method used as a screening test for bacterial infection detection. Conventional isolation method for isolating and identifying Pseudomonas aeruginosa required 24-96 hours. As a result, crucial time in the management of inflicted patient especially in cases with severe sepsis lead to empirical use of antibiotics which is inappropriate in high number of the cases has led to the emerge of multidrug resistant pathogens and threatened patient life's. Current methods for rapid identification of pseudomonas require advanced automated machine to be operated by highly skilled technicians.
Nanotechnology with magnetic nano and microparticles have received significant attention in recent two decades. They provide broad opportunities for application in chemistry, biochemistry, biology, and medicine. The magnetic nanoparticles (MNPs) have a wide range of highly specific surface area and controlled under the external magnetic field, with high sensitivity, high specificity, low back ground and easy for quantitative analysis. The combined use of magnetic particles technology for the development of efficient biosensors for clinical purposes as diagnostic tool is emerging trends (Tang Y et al., 2014).
Staphylococcus aureus (S. aureus)
Staphylococcus aureus (S. aureus) is a facultative anaerobic, gram-positive bacterium discovered by Dr. Alexander Ogston in 1880 (Ogston A, 1984). In recent years, S. aureus received a lot of attention as it is among the top five common pathogens associated with food-borne illnesses nationwide and one of the most commonly isolated pathogens in hospital-acquired infections (Swartz M N, 1994) and healthcare facilities (Wang C H et al., 2011). S. aureus is found in the nostrils and on the skin of warm-blooded animals, including humans and thus can contaminate food products that are derived from animals such as meat, milk and eggs (Yang H, 2011). Also poor hygiene by food handlers during processing and preparation could also contaminate foods (Goto M et al., 2007). What's more, S. aureus could live in harsh environments. Thus, under temperature-abused conditions, it can grow and produces enteric toxins. These enterotoxins are heat stable and resistant to the processing and normal cooking temperatures, which usually inactivates or kills the bacterial cells and so causes staphylococcal food poisoning (SFP) (Doyle M P et al., 2007) which is marked by severe gastrointestinal symptoms such as emesis, diarrhea, and/or abdominal pain after a four hour incubation period. Recently, the Center for Disease Control and Prevention (CDC) estimates 240,000 illnesses with 1,000 hospitalizations and 6 deaths associated with staphylococcal food poisoning annually (Scallan E et al., 2011).
S. aureus usually colonizeopen wounds and urinary tracts leading to numerous illnesses, from minor skin infections to life-threatening diseases, such as abscesses (Kapral F A et al., 1980), pneumonia (Robertson L et al., 1958), meningitis (Gordon J J et al., 1985), endocarditis (Fowler V G et al., 2007) and septicemia Cross A S et al., 1983). The National Institutes of Health and Centers for Disease Control and Prevention reported 94,000 life-threatening antibiotic-resistant infection cases out of 500,000 people infected with S. aureus in United States of America annually (Klein E et al., 2007).
Conventionally, S. aureus detection and identification is based on bacterial culture methodology (Bocher S et al., 2008). However, this process is time-consuming, labor-intensive and can take up to several days for identification of the pathogenic bacteria which is an unacceptable delay in emergency and critically illness situations such as sepsis. Thus, this protocol always limits its practical application for rapid clinical diagnosis (Gilbert G L et al., 2002). Other ultra-sensitive detection methods are based on nucleic acid amplification, such as polymerase chain reaction (PCR) (Cheng J C et al., 2006), ligase chain reaction (LCR) (Moore D F et al., 1998) and strand displacement amplification (SDA) (Edman C F et al., 2000) have been employed. Fortunately, these technologies are capable of detecting low numbers of bacterial cells but within several hours. Moreover, these technologies are expensive and require complex procedures such as, prior bacterial DNA isolation, preparation of enzyme reaction mix and expensive instruments for nucleic acid amplification. Accordingly, their use in clinical diagnosis is limited. Other alternative detection methods such as antibody-based immunoassays were well established and have been used Swaminathan B et al., 1994). However, since antibodies are proteins which cannot be amplified, ultrasensitive detection is limited. Nevertheless, this limitation was circumvented by the development of immuno-PCR assay. In this technology, antibody is cross-linked with DNA “barcode” for PCR amplification (Huang S H et al., 2004). However, antibody-DNA complexes conjugation and purification is a daunting task. In addition to the urgent need for expensive instruments. Notomi T et al. (2000), developed a loop-mediated isothermal amplification (LAMP) assay targeting the arcC gene of S. Aureus (Lim K T et al., 2013). This assay was equally specific to PCR with a shorter detection time. Recently, Chang Y C et al. (2013) reported the development of a non-PCR-based method which combines aptamer-conjugated gold nanoparticles and a resonance light-scattering detection system. This method successfully detects a single S. aureus cell within 1.5 hours. Notably, none of the above mentioned detection methods fully satisfies the detection performance criteria since they are sophisticated, costly in terms of time and money and involves burdensome preparatory steps and sophisticated instruments. Therefore, there is a great need to develop a “sample-to-answer” highly specific and sensitive detection and diagnostic method that can be performed within a short period.
Escherichia coli (E. coli)
In recent years, outbreaks of foodborne diseases associated with pathogenic E. coli have widely spread and grown as apublic health problem. Traditional and currentdetection techniques of food microbial pathogensare time-consuming, require expensive instrumentation and are labor intensive.
Food borne illness linked to the consumption of fresh and minimally processed food cause a myriad of discomfort and economic loss for many people each year (Shriver-Lake et al., 2007; Beuchat L R et al., 2002; Tauxe R et al., 1997). In general, washing fresh fruits and vegetables with cold water removes lingering dirt present on the surface but does not remove pathogens that may exist inside. Fresh products might be contaminated with microbial pathogens upon contact with contaminated water or manure from the soil (Ingham S C et al., 2004; Song I et al., 2006). A current report has showed that the United States Department of Food Safety and Inspection Services (FSIS) spend over half a billion dollars annually on food inspections for bacterial contaminants. As approximately 73000 food borne infections cases occur every year, out of which 2-7% of these cases suffers from a severe complication called hemolytic uremic syndrome (HUS) (Griffin D W et al., 2003).
Obviously, improvements of food borne pathogens identification techniques after ingestion are important for treatment, but it is more important to prevent infections. One way of doing so is to identify contaminated food products prior to ingestion, preferably before it is distributed to grocery stores, restaurants and manufacturing facilities. This could be achieved through the development of a detection device applicable at the retail level to protect the consumer.
Today, several conventional techniques are used to detect pathogenic microbes: culturing method is commonly used but remain problematic due to the lack of phenotypic characteristics which distinguish between generic pathogens (Gould G et al., 2009), DNA-based assays are currently the most specific and sensitive test available as a confirmatory assay (Uttamchandani M et al., 2009; Call D et al., 2005; Simpson J M et al., 2005; Deisingh A K et al., 2004). However, in these assays long time (up to 48 h) is required to obtain results due to the extensive sample pretreatment steps including enrichment and extraction. Also, highly trained staff are required to perform the assay and analyze the results. Moreover, polymerase chain reaction (PCR) inhibitors such as humics are commonly presented in complex food matrices and must be removed prior to analysis (Shriver-lake L C et al., 2007). Other immunoassay-based methods have been recently employed to achieve lower levels of sensitivity while avoiding many of the disadvantages of the DNA-based assays.
However, these methods are less specific than DNA assays (Shriver-lake L C et al., 2007). Presently, researchers are attempting to improve the specificity of these assays through the employment of antibodies and the use of changes in optical properties such as the transmitted light, surface plasmon or acoustic waves resulting from antibody-antigen binding (Deisingh A K et al., 2004; Subramania A et al., 2006; Berkenpas E et al., 2006). Additionally, some of these assays used fluorescent labels to provide an optical signal (Nyquist-Battie C et al., 2005; Ho J A A et al., 2003). However, limitations of these assays are related to the inconsistency and high variability of target DNA labeling. In addition, they utilize expensive and nonportable scanners for data acquisition and analysis (Call D R et al., 2005; Kuck L R et al., 2008; Vora G J et al., 2008). Other alternative detection methods such as enzyme-linked immunosorbent assay (ELISA) (Abuknesha R A et al., 2005), spectrometric (Siripatrawan U et al., 2007) and electrochemical were also employed (Guo Y et al., 2015) for the detection of food borne pathogens. With these methods a detection range of 103 to 105 cells mL−1 was achieved without enrichment and was as low as 1 CFUmL−1 with enrichment. Nevertheless, this low limit of detection is valid only for the detection of microorganism that can be grown on specific media. Kuck L R et al. (2008) have illustrated a colorimetric assay for bacterial detection. This assay used unstable reagents that require temperature-controlled environments with a variable development times, leading to an increase in nonspecific background (Kuck L R et al., 2008). Notably, none of the above mentioned detection methods fully satisfy the detection performance criteria since they are sophisticated, costly in terms of both time and money and requires burdensome preparation steps.
Porphyromonas gingivalis (P. gingivalis)
Periodontal diseases are inflammatory diseases of microbial etiology affecting the hard and soft supporting tissues of the teeth (Feng Z M et al., 2006). The term “periodontal disease” encompasses two subclasses, gingivitis and periodontitis. Gingivitis is characterized by the inflammation of the gums without loss of connective tissue attachment or bone. Gingivitis is a prerequisite for, but does not necessarily lead to periodontitis (Armitage G et al., 1997; Listgarten M et al., 1980; Fowler C F et al., 1982; Kretschmar S et al., 2012) which affects approximately 7-15% of the adults in the western world, making it one of the most common diseases (Bostanci N et al., 2012). Moreover, epidemiological and mechanistic evidence has linked periodontitis to other systemic illness such as atherosclerosis, cardiovascular diseases and rheumatoid arthritis (Zhang B et al., 2013; Darveau R P et al., 2010; Kebschull M et al., 2010; Ogrendik M et al., 2013).
The microbiota of the human oral mucosa consists of a myriad of bacterial species that normally exist in commensal harmony with the host (Mysak J et al., 2014). However, studies have implicated a specific bacterial group, named as “red complex” and including Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola as the causative agents of periodontal diseases (Haffajee A D et al, 1983).
In particular, P. gingivalis is an opportunistic pathogen, intensively participates in the initiation and progression of periodontal disease. It is a gram negative, assacharolytic, black-pigmented species which colonizes the subgingival region (Varghese J et al., 2013). It invades tooth supporting tissues and evade the host defense mechanisms causing periodontitis (O'Brien-Simpson N M et al., 2004). It is armed with a pleothera of virulence factors such as lipopolysaccharide (LPS), gingipains, peptidyl arginine deiminase, haemagglutinins, fimbriae and outer membrane proteins (Bostanci N et al., 2012; Haffajee A D et al., 1983; Holt S C et al., 1999; Lamont R J et al., 1998; Robertson P B et al., 1982). These virulence products assist its survival in periodontal pocket and contribute to the destruction of the tooth's supportive tissues. Also, these products weaken hosts' defense system during the periods of elevated bacterial activity. Gingipains, which are typsin-like cysteine proteases account for at least 85% of the general proteolytic activity displayed by P. gingivalis (Kaman W E et al., 2012; Haffajee A D et al., 1983). The current understanding of periodontitis pathogenesis suggests that gingipain proteases have an important role in the disease onset and progression (Kaman W E et al., 2012).
Up to date, many methods have been developed to assess periodontal diseases. Some of these methods include subjective observational indices which are based on criteria such as bleeding on gentle probing, pocket depth, attachment loss and radiographic evidence of bone loss. Of these indicators, only bleeding on probing has been claimed to correlate with active periodontal disease. Nevertheless, bleeding itself is a subjective indicator of disease and the diagnostic value of bleeding on probing has been questioned, as such bleeding appears to be allied with a high proportion of false positive indications of periodontal disease (Haffajee A D et al., 1983). Moreover, this method does not identify the causative agents (Kaman W E et al., 2012). An alternative diagnostic methods capable of identifying the periodontal pathogens include culture-based, nucleic acid-based and antibody-based assays (Choe Y et al., 2006; Kuboniwa M et al., 2003; Jervoe-Storm P M et al., 2010). However, these methods were very laborious and time consuming. So far, direct detection and identification of periodontal pathogens in situ have proven difficult (Kaman W E et al., 2012). Furthermore, Loesche and coworkers described a diagnostic test based on the enzymatic diagnosis of periodontal pathogens (Loesche W J et al., 2010). However, these test required the use of sophisticated instrument and a trained personnel. Currently, diagnostic tests are based on the measurement of a specific component in the cervicular fluid or the measurement of cerevicular fluid volume. This was based on the fact that gingivitis and periodontitis were characterized by accumulation and flow of cervicular fluid (a transudate of serum) at the gingival sulcus and pockets.
Notably, a clear lack of biochemical markers useful for the detection of current and future periodontal disease activity in the cervicular fluid prompted researchers to look for a specific chemical compound, mainly a protein, such as an enzyme or a cytokine, in fluids from the oral cavity of a patient, such as gingival cervicular fluid (GCF) to successfully and specifically diagnose periodontal diseases (Mailhot J M et al., 1998) and ultimately aid in the design of more effective therapies.
Listeria monocytogens (L. monocytogens)
Listeria is a gram positive, rod-shaped, non-spore-forming facultative anaerobic bacteria consisting of six species: L. monocytogenes, L. innocua, L. seeligeri, L. welshimeri, L. ivanovii, and L. grayi. Out of which, L. monocytogenes species is commonly associated with human listeriosis.
Listeriosis is a disease manifested by fever and muscles ache. It could be either in an invasive or noninvasive form. The invasive form can spread to the central nerves system causing headache, stiff neck, and confusion, loss of balance, convulsion and ocular listeriosis (Mead P S et al., 2006). Also, listeriosis is highly common among pregnant women (Centers for Disease Control and Prevention, 2013), elderly patient above 65 year (CDC, 2011) and in immunocompromised patients (Bala B, 2007). I n pregnant women listeriosis causes miscarriage, stillbirth, perinatal septicemia and meningitis in new born baby (Mokta et al., 2010).
As an issue of concern, L. monocytogenes is considered a major source of human foodborne illness worldwide due to its presence in the ready to eat food (Roberts T et al., 2009; Vazquez-Boland J A et al., 2001; Garrido V et al., 2008; O'Connor L et al., 2010; Junttila J R et al., 1988; Liu D, 2006). At present, the United States Department of Food Safety and Inspection Services (FSIS) spent billions of dollars for inspection of bacterial contamination annually (Centers for Disease Control and Prevention, 2013). Listeria has been widely observed in environmental samples such as water, soil, silage and in food samples such as dairy product (milk, soft cheese), meat, and sea food (cooked and raw). This microbial contaminant can tolerate high environmental stress form and a wide range of temperature (−18 to 10° C.), being a psychrophillic organism.
In food industry the time between food packaging, inspection and consumption is crucial as small undetectable number of microbes at the time of packing can multiply and become life threatening by the time of consumption. However, L. monocytogenes detection in foods is hampered by certain limitation as the high population of competitive microflora, the low levels of the pathogen and the interference of inhibitory food components (Norton D M, 2002).
Typically, L. monocytogenes detection and identification in food involve standard culturing technique which requires trained personnel and long time as negative results can be confirmed in 3-4 days. Whereas, positive results might take 5-7 days (Jadhav S et al., 2012; Alessandria V et al., 2010; Kabuki D Y et al., 2004; Frece J et al., 2010; Gasanov U et al., 2005; Brehm-Stecher B F et al., 2007). Notably, in food industry, it is not common to hold food products for 7 days prior to distribution. Moreover, the standard culture method requiring selective enrichment with subsequent culturing on selective media, followed by serological and/or biochemical tests (Wang D et al., 2011). Later on, alternativether method based on specific antigen-antibody reaction was developed, but this immuno assay method was less sensitive than culturing method with a lower limit of detection (LOD) of 10τ-10υ cells/ml (Gasanov U et al., 2005). Moreover, antibody preparation is time consuming with a chance of detecting false positive (Zhang D et al., 2009). Other method based on nanoparticle immuno magnetic separation and real-time Polymerase Chain Reaction (PCR) was able to de lect 102 CFU/0.5 mL of L. monocytogens in milk (Yang H et al., 2007). However, this method is limited by the presence of PCR inhibitors in the real biological samples and food samples. Therefore, it is necessary to develop a rapid, sensitive, and cost-effective method for microbiological examination of real food samples.
Listeria has several virulence factor which contributes to its pathogenicity. During the infection, L. monocytogenes invade the host cell by lysing the phagocytic vacuoles and proliferate in the cytosol with the help of Listeriolysin O (LLO) and two secreted phospholipases C (PLC): a broad-range phospholipase C (PC-PLC) and a phosphatidylinositol specific PLC (PI-PLC) (Portnoy D A et al., 1992; Smith G A et al., 1995). Broad-range phospholipase C is released in an inactive propeptide and it is activated by the cleavage of the propeptide by a metalloprotease (Mpl) at low pH. 15. Accordingly, higher level of zinc dependent-metallo protease would be observed in the host infected with L. monocytogens. This virulence protease capable of cleaving specific substrate (Mitchell C S et al., 2003; Kasana R C et al., 2011) could be used as a biomarker for the detection of L. monocytogens food contamination. Remarkably, Mitchell C S et al. (2003), pronounced a specific peptide substrate which could be selectively cleaved by L. monocytogens protease.
Accordingly, there is still a need to develop simple, sensitive, specific, rapid, cost-effective colorimetric bio sensors capable of detecting the presence or absence of a pathogenic microorganism in a sample upon suspicion. The sample may be related to food products, hospitals, health centers or any other environment.